Fig. 1: Identification of neoantigenic epitopes in the B16F10 melanoma model.

a Schematic (made using Biorender with full license) depicting B16F10 neoantigen identification. b C57BL/6 mice (n = 5 independent biologic replicates) were immunised with peptide vaccine targeting putative B16F10 neoantigens. Seven days post-immunization splenocyte-derived T cells were stimulated with mutant peptide (solubilised in DMSO) for 6 h then IFNγ production was measured by flow cytometry. Symbol indicates individual mice (n = 5/condition), error bars indicate the group median, ±95% confidence interval (CI). Solid line indicates assay lower limit of detection (LLD). Dashed indicates upper limit of 95% CI for negative responses. Statistical analysis: one-way Kruskal–Wallis test, followed by Dunn’s test for multiple comparisons, with alpha level set to 0.05. Colour key: red; T cell-elicited response significantly above the LLD, grey; insignificant T cell response, black; T cells not stimulated with any peptide (DMSO only control). Where p < 0.001, p value was too low for Prism software to provide an exact value. c C57BL/6 mice (n = 4/group, repeated four times) were immunised with (long) peptide vaccine targeting putative B16F10 neoantigens. Seven (7) days post-immunization, flow cytometry was performed on splenocyte-derived CD8⁺ T cells. Flow cytometry plots are organized in columns; data from vaccinated mice (left) vs unvaccinated (right). Representative tetramer staining is shown. Gating strategy shown in Supplementary Fig. 2. d As in (b). Error bars depict the group median, ±95% confidence interval (CI); n = 5 independent biologic replicates. Peptide stimulation of T cells by tumour-associated antigens (TAAs) [blue], (non-tumour) OVA antigen [dark grey], neoantigens eliciting measurable tetramer response [red], neoantigens not eliciting measurable tetramer response [light grey]. e Attributes of neoantigens (top) predicted computationally and TAAs previously characterised^40,41,42. Binding affinity (BA)-Rank values are shown for mutated (ALT) and wild type (REF) peptides. Table subscript legend is as follows: ¹NetMHCpan v.4.1 BA-Rank% value. ²Variant a.a. position vis-à-vis learned FlexPepDock structure. f In silico MHC-I binding affinity analysis of Hsf2 p.K72N_68-76 demonstrates high affinity binding and high differential binding affinity between ALT and REF peptides, hereafter referred to as mutant (MT) and wild type (WT) peptides, respectively. Source data are provided as a Source Data file.